The essential oil of peppermint (Mentha×piperita) is composed principally of C3-oxygenated p-menthane monoterpenes, and the characteristic organoleptic properties of this oil are derived predominantly from (−)-menthol. Menthol biosynthesis begins with the diversion of the primary isoprenoid intermediates, isopentenyl diphosphate and its allylic isomer, dimethylallyl diphosphate, to geranyl diphosphate (the precursor of all monoterpenes) by the prenyltransferase geranyl diphosphate synthase (GPPS), and is followed by ionization and cyclization of this C10 intermediate to limonene by the terpene cyclase (−)-(4S)-limonene synthase. Regiospecific and stereospecific hydroxylation of this monoterpene olefin intermediate is catalyzed by cytochrome P450 limonene-3-hydroxylase to yield (−)-trans-isopiperitenol, and is followed by dehydrogenation of this allylic alcohol (by isopiperitenol dehydrogenase) to afford the α,β-unsaturated ketone (−)-isopiperitenone. The endocyclic double bond of this intermediate is reduced by isopiperitenone reductase to yield (+)-cis-isopulegone, which undergoes enzymatic isomerization of the isopropenyl double bond (Δ8,9) to yield the conjugated ketone (+)-pulegone (Δ4,8). The newly formed isopropylidene double bond is reduced by pulegone reductase to yield (−)-menthone and lesser amounts of (+)-isomenthone. Alternatively, in a cytochrome P450-mediated side-reaction, pulegone undergoes C9 hydroxylation and intramolecular cyclization and dehydration to yield (+)-menthofuran. In the final reductive step of the pathway, (−)-menthone and (+)-isomenthone are reduced to (−)-menthol and (+)-neoisomenthol, respectively, or, by a separate reductase, to (+)-neomenthol and (+)-isomenthol, respectively; the latter three monoterpenol isomers are minor constituents of peppermint oil.
Monoterpene biosynthesis in peppermint occurs in the highly specialized secretory cells of epidermal oil glands, and recent evidence suggests this metabolic process is divided into two temporally distinct periods of transcriptional and translational activity. The initial process is characterized by the de novo biosynthesis of the p-menthane monoterpenes (as determined by 14CO2 incorporation) which results in the accumulation of mostly (−)-menthone. In vitro assay of the relevant enzymes of menthone biosynthesis demonstrated that these activities appear coincidentally during leaf expansion, and endure for a brief time period (12-20 days post leaf initiation) with peak activity levels correlating with the essential oil secretion (gland filling) phase of gland development. RNA-blot analysis further showed that maximum transcript accumulation of limonene synthase occurs immediately prior to maximal enzyme activity, suggesting that at least the first committed enzyme of monoterpene biosynthesis is transcriptionally regulated. However, the production of (−)-menthol from menthone is not significant until late in leaf development, after de novo monoterpene biosynthesis is essentially complete; this process occurs in mature oil gland cells during the post-secretory phase. This second developmental process, termed “oil maturation”, is characterized by the depletion of the dominant intermediate menthone, and is concomitant with increased activity of the menthone reductases and the accumulation of menthol (and lesser amounts of the epimer neomenthol).
The present invention provides isolated nucleic acid molecules (e.g., cDNA molecules) that encode menthone: (3R)-(−)-menthol reductase that converts (−)-menthone to (3R)-(−)-menthol. The present invention also provides isolated menthone: (3R)-(−)-menthol reductase proteins.